1. Field of the Invention
This invention relates to transport vectors for targeting drugs to cells in vitro and to tissues in vivo. In particular, this invention relates to targeting vectors based on avidin-biotin technology for delivery of peptides and oligonucleotides to cells and tissues in vivo and in vitro. The invention further concerns soluble transport vectors that are comprised of avidin fusion proteins which mediate cellular uptake of biotinylated oligonucleotides and peptides, as well as avidin protection of circulating oligonucleotides from serum 3'-exonucleases.
2. Description of Related Art
The publications and other reference materials referred to herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference. For convenience, the reference materials are numerically referenced and grouped in the appended bibliography.
Antisense oligonucleotides are potentially highly specific chemotherapeutic agents for the treatment of cancer, viral infections, and other disorders (1). There are two principal obstacles to effective delivery of antisense oligonucleotides to tissues: (a) poor transport of oligonucleotides across cell membranes, and (b) rapid degradation by tissue and serum nucleases. The minimal cellular uptake of the highly charged oligonucleotide compounds has been dealt with by adding to cells in tissue culture concentrations of antisense oligonucleotides ranging from 10 to 100 .mu.M to achieve biological effects (3). Prohibitively higher concentrations will be required in vivo where capillary barriers retard the delivery of oligonucleotides to cells.
The nuclease problem has been dealt with by synthetic preparations of phosphorothioate nucleic acid derivatives (4). However, these nucleic acids require the use of unnatural nucleotides and thus cannot be prepared by recombinant DNA technology. The necessity for organic synthesis of the phosphorothioate oligonucleotide may significantly limit the industrial scale production of such compounds.
Liposomes have been used to deliver antisense oligonucleotides to tissues in vitro, in particular where capillary barriers are absent (5). However, liposomes are not effective delivery vehicles in vivo because they selectively deliver the drug to macrophages lining the reticuloendothelial system, and because they are too large to effectively cross capillary barriers in vivo (6). In particular, liposomes have not proven to be effective drug delivery vehicles for transport into brain across the brain capillary barrier system, i.e., the blood brain barrier (BBB) (7).
Recognizing this problem, other investigators have prepared polylysine conjugates with vector proteins such as asialofetuin, which is taken up by receptor mediated endocytosis into liver cells, or transferrin, which is taken up by organs expressing high quantities of transferrin receptor on the cell membrane (8, 9). The limitations of this approach are two-fold.
First, the asialofetuin- or transferrin-polylysine conjugate must be prepared chemically, oftentimes with low yields. Secondly, the interaction between the polylysine and the antisense oligonucleotide is not covalent and subject to rapid dissociation in vivo. Therefore, it would be advantageous to conjugate the antisense oligonucleotide to the transport vector via a high affinity bond that is stable in circulation but is labile in tissues.
It would be desirable to eliminate the need to couple the DNA binding protein to a vector compound. It would also be desirable if the targeting molecule to which the nucleotide binds would be stable in the circulation in vivo. It would be further desirable if such a targeted oligonucleotide was resistant to serum nucleases, thereby avoiding the need to use synthetic and unnatural oligonucleotides in preparation of antisense derivatives.
Current peptide delivery to tissues entails the use of liposomes (10), which have the limitation described above, and the preparation of chimeric peptides (11). The latter involves covalent conjugation, generally using disulfide bonds, of the pharmaceutical peptide to its transport vector.
This often involves complex linker chemistry. It would be desirable if peptide drug delivery could be achieved with simpler linker chemistry to yield linkage of a peptide to a transport vector, the linkage being of high affinity and in high yield. It would be further desirable if the link between the peptide and transport vector would be stable in plasma and labile in cells.
Peptide delivery to tissues in vivo involves the formation of chimeric peptides by covalent bonding of peptides to transport vectors. (11) The complex linker chemistry involved in the formation of chimeric peptides often produces low chemical yields which may not be optimal for industrial-scale production. It would be desirable to have linker chemistry in peptide delivery that is simple, associated with high yields and may be applied to industrial-scale production.
Accordingly, further developments are needed to make available a conjugation chemistry that would allow for tight binding of a drug, such as an oligonucleotide or peptide, to a transport vector within the circulation. It would be desirable to eliminate linker chemistry that involves covalent attachment of oligonucleotide or peptide drug to a tissue specific transport vector (12). Further desired would be a linker technology for drug delivery which provides the advantages of stability in plasma, lability in tissues, and high efficiency of drug/vector coupling that is necessary for industrial-scale production of chimeric peptides or oligonucleotides.